research open access nebulized perflubron and carbon

El Mays et al. Respiratory Research 2014, 15:98http://respiratory-research.com/content/15/1/98

RESEARCH Open Access

Nebulized perflubron and carbon dioxide rapidlydilate constricted airways in an ovine model ofallergic asthmaTamer Y El Mays1, Parichita Choudhury1, Richard Leigh1, Emmanuel Koumoundouros2, Joanne Van der Velden2,3,Grishma Shrestha1, Cora A Pieron4, John H Dennis4, Francis HY Green1,5* and Ken J Snibson2

Abstract

Background: The low toxicity of perfluorocarbons (PFCs), their high affinity for respiratory gases and their compatibilitywith lung surfactant have made them useful candidates for treating respiratory diseases such as adult respiratory distresssyndrome. We report results for treating acute allergic and non-allergic bronchoconstriction in sheep using S-1226 (a gasmixture containing carbon dioxide and small volumes of nebulized perflubron). The carbon dioxide, which is highlysoluble in perflubron, was used to relax airway smooth muscle.

Methods: Sheep previously sensitized to house dust mite (HDM) were challenged with HDM aerosols to induce earlyasthmatic responses. At the maximal responses (characterised by an increase in lung resistance), the sheep were eithernot treated or treated with one of the following; nebulized S-1226 (perflubron + 12% CO2), nebulized perflubron +medicalair, 12% CO2, salbutamol or medical air. Lung resistance was monitored for up to 20 minutes after cessation of treatment.In additional naïve sheep, a segmental bronchus was pre-contracted with methacholine (MCh) and treated with nebulizedS-1226 administered via a bronchoscope catheter. Subsequent bronchodilatation was monitored by real time digital videorecording.

Results: Treatment with S-1226 for 2 minutes following HDM challenge resulted in a more rapid, more profound andmore prolonged decline in lung resistance compared with the other treatment interventions. Video bronchoscopyshowed an immediate and complete (within 5 seconds) re-opening of MCh-constricted airways following treatment withS-1226.

Conclusions: S-1226 is a potent and rapid formulation for re-opening constricted airways. Its mechanism(s) of action areunknown. The formulation has potential as a rescue treatment for acute severe asthma.

Keywords: Asthma, Novel treatments, Sheep model, Allergic airway disease, Carbon dioxide, Perflubron

BackgroundAsthma is a common chronic disorder characterized byairway hyper-responsiveness, bronchial smooth musclehypertrophy, increased mucus secretion, and airway in-flammation [1,2] on a background of airway wall remod-elling and mucous plugs [3,4]. Asthma is triggered bymany factors such as allergens (e.g., pollen, pet dander),irritants (e.g., smoke), drugs, cold, exercise [5], and

* Correspondence: [email protected] Inflammation Research Group, Snyder Institute for Chronic Diseases,University of Calgary, Calgary, AB, Canada5Department of Pathology & Laboratory Medicine, HRIC 4AC62 3280 HospitalDrive N.W, Calgary, AB T2N 4Z6, CanadaFull list of author information is available at the end of the article

© 2014 El Mays et al.; licensee BioMed CentraCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

upper respiratory tract infections [6]. Based on recenttrends, the worldwide prevalence of asthma mayincrease from 300 million individuals currently to 400million individuals by 2025 [7]. Healthcare costs ofasthma are considered to be among the highest associ-ated with chronic disease [8,9]. In Canada, 59% ofasthma patients treated in general practice are estimatedto have uncontrolled disease [10]. Inhaled short actingβ2-agonists represent the most common front-lineemergency department treatment for acute exacerba-tions due to their well-known rapid bronchodilatory ef-fect [11–13] β2-agonists, while generally considered safemay have significant side effects [14] and approximately

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one fifth to one third of patients do not respond tofront-line treatment with short acting β2-agonists andultimately require hospitalisation [15]. Furthermore, aclinically significant proportion of asthma patients arerefractory to conventional treatments, posing a chal-lenge for their management [16]. A new class of bron-chodilators working through a different mechanismwould be a useful addition for treating acute asthma.In acute severe asthma, conventional treatment with in-

haled β2 agonists may no longer be effective [11–13], asthe therapeutic aerosol may be unable to penetrate air-ways obstructed by mucous plugs [4], increasing the riskof sudden death [17]. Emergency treatment for asthma in-cludes mechanical ventilation [15] which may cause injury(barotrauma) to the ventilated segments of the lung [18].Hence, a rapid non-invasive treatment for severe, life-threatening asthma is needed.Inhaled carbon dioxide is a known bronchodilator for

many mammalian species [19]. Hypercapnia has long beenknown to relax airway smooth muscle in vitro [20,21],whereas hypocapnia causes airway smooth muscle con-traction [22]. Hyperventilation is a well known trigger ofasthma [23]. Studies by Fisher et al. [24] and Mc Faddenet al. [25] indicate that low alveolar PCO2 (PACO2) maybe associated with an increase in airway tone in asthmaticpatients. By contrast hypercapnia (induced by a short cir-cuiting CO2 absorber) has been shown to reduce airwayresistance in both healthy and asthmatic subjects [26]. Fi-nally a study reported by Fisher and Hansen [27] showedthat inhalation of 6% CO2 over 4–5 minutes relieved exer-cise induced airflow obstruction in young atopic asth-matics at rest and during exercise.Perfluorooctylbromide (perflubron) is a chemically

inert perfluorocarbon (PFC). It has a high solubility forrespiratory gases such as CO2 and O2 and a low surfacetension compatible with endogenous airway surfactant[28–30]. It has been used in total and partial liquidventilation studies for treatment of ARDS [28]. Inaddition, perflubron has been shown to reduce pul-monary inflammation and injury through a variety ofactions [28,31–33]. The high density and low surfacetension of perflubron should also facilitate the penetra-tion of obstructed airways and re-expansion of col-lapsed lung [34].There are no reported studies of PFC use in asthma.

However, its properties of gas transport for CO2 and O2,low interfacial tension at the PFC-mucus interface, lowtoxicity and anti-inflammatory properties [31–33,35]make it a good candidate as a rescue therapy in lifethreatening acute asthma. Recently perflubron aerosolshave been used to successfully treat acute lung injury inan animal model [36,37].The major challenge for treating acute severe asthma

is to open constricted airways rapidly enough to allow

delivery of conventional medication to the diseasedairways.In this study we show that inhaled nebulized perflu-

bron + CO2 (S-1226) rapidly opens constricted airwaysin an ovine model of allergic asthma using house dustmite as allergen [38,39].

MethodsExperimental sheepFemale merino-cross farm-reared sheep aged 6 monthswere used in the experiments described here. After thesensitisation procedures (described below), sheep weretransported to Animal House Facility at Veterinary Sci-ence, Parkville, Australia where they were kept in indoorpens for all the procedures described in this report. Onarrival at the Animal House Facility all sheep weretreated with an antihelminthic to ensure that they wereparasite-free.All experimental protocols used in this study were ap-

proved by the Animal Experimentation Ethics Commit-tee at the University of Melbourne.

Drug sourceAcetyl-β-methylcholine chloride (MCh) was from Sigma-Aldrich, Stenhein, Germany and salbutamol sulphate solu-tion was purchased from GSK Pty Ltd Australia. Gas mix-ture (12% CO2 + 21% O2+ balance N2) was from CIG PtyLtd Australia.

Characterization of nebulized perflubronCharacterization of perflubron delivered by the Sidestreamnebulizer was conducted under cGMP conditions atSolAero Ltd laboratory. Methods to characterize perflu-bron aerosol were adapted from regulatory methods em-bodied in the European Nebulizer Standard [40]. Theunique nature of perflubron required adaptation ofmethods including incorporation of trace quantities (5%)of a perflubron compatible non-volatile PFC which couldbe assayed gravimetrically to determine both aerosol out-put as well as particle size distributions. Aerosol outputwas determined using a breath simulation (500 mL at15 cycles per minute) and collecting released aerosolrepresenting the ‘inhaled aerosol’ on a pre-weighed 3Melectrostatic filter, subsequently allowing volatile perflu-bron to dissipate and quantitatively weighing the non-volatile PFC component. The residual weight gain wasthen used to back calculate the original amount of depos-ited aerosol. Pre- and Post- weights of nebulizers providedthe gross perflubron emitted, from which the analyzedaerosol proportion could be subtracted to estimate theproportion of perflubron in the vapor phase. Aerosol sizewas determined using a Marple 298X cascade impactorequipped with pre-weighed GF/A filters from which the


non-volatile aerosol residue was similarly gravimetricallydetermined to quantify size fractions.

House dust mite (HDM) sensitization and airwaychallengesSheep 6 months of age were subcutaneously sensitized with50 μg house dust mite (HDM) extract (Dermatophagoidespteronyssinus; CSL Ltd, Parkville, VIC, Australia) mixed 1:1in aluminum hydroxide [Al(OH)3] adjuvant. Injections of1 ml HDM / Al(OH)3 were given on three occasions at2 week intervals [38]. Levels of HDM-specific IgE (IgE-HDM) were determined by enzyme-linked immunosorbentassay (ELISA) of serum samples obtained from animals im-mediately prior to, and 7 days after, HDM sensitization[38,41]. Sheep were only selected for use in the study if theyhad increased levels of HDM-specific IgE (IgE-HDM) ≥2-fold above pre-sensitization values.To induce pulmonary sensitization to HDM, all sheep

were given three inhalation challenges of aerosolisedHDM (1 mg/ml in 5 ml of phosphate buffered saline).Lung function was recorded for one hour directly after theaerosol HDM challenge. HDM aerosols were generatedwith a medical nebulizer (Vitalair RapidNeb, Allersearch,Vermont, Australia). The nebulizer was connected to adosimeter system consisting of a solenoid valve and asource of compressed air (20 psi). The output of the nebu-lizers was directed into a T-piece with one arm connectedto the inspiratory port of a mechanical ventilator (IntermedBear 2 – Adult Volume Ventilator, Bear Medical Systems,Inc., Riverside, CA). HDM challenges were delivered at atidal volume of 500 mL and a rate of 20 breaths per minutefor 10 minutes.

Measurement of lung functionData on lung function were obtained from all sheep usingestablished techniques [42]. Briefly, pressures and flowwere acquired from conscious unsedated sheep using aNational Instruments Data Acquisition (DAQ) systemwith a Virtual Instrument programmed with Lab View®(National Instruments, Austin, USA). The sheep were re-strained in a body sheath and head restraining harnesstethered to a custom-made animal trolley. To measureoesophageal pressures, a balloon pressure catheter was ad-vanced through one nostril into the lower esophagus. Toassess tracheal pressures throughout the respiratory cyclea cuffed endotracheal tube was placed into the tracheaand then a side-hole catheter was advanced 2 cm past thedistal end of the endotracheal tube. The oesophageal andtracheal catheters were connected to differential transduc-ers (Gaeltec 8 T, Gaeltec Ltd, Dunvegan, Scotland) tomeasure oesophageal pressure (Poes) and airway pressures(Paw) respectively. Transpulmonary pressure (Pl) was ob-tained from Poes with respect to Paw. Airflow was mea-sured via a pneumotachograph (Hans Rudolph Inc,

Kansas City, USA) attached to the proximal end of theendotracheal tube. Calibration and synchronization of thegas transducers and pneumotachograph were performedas described previously [43,44]. Lung dynamics were de-termined by a modified Mead-Whittenberger method[45]. Breath by breath analysis of flow and pleural pressure(Ppl) was used to determine lung resistance (RL) from anaverage of five breaths. Data from breaths which differedby more than 20% in volume and more than 10% of thebreathing rate were rejected. Acquisition and detailed ana-lysis of pulmonary physiological data were performed oncustomized Lab View software developed by Koumoun-douros et al. [42].

Treatment deliveryTreatments with nebulized S-1226, nebulized perflubronand nebulized salbutamol were generated with a Side-stream® nebulizer (Philips Respironics). The nebulizerwas connected to a dosimeter system consisting of a so-lenoid valve and a source of compressed (20 psi drivingpressure) gas mixture (12% CO2, 21% O2 and 67% N2)for S-1226 or compressed medical air (21% O2 and79% N2) for perflubron and salbutamol. Treatmentswere delivered at a tidal volume of 500 mL and a rate of20 breaths per minute for 2 minutes. Treatments withCO2 alone and medical air were performed following thesame protocol but with an empty nebulizer.

HDM challenge followed by treatmentSheep (previously HDM sensitized) were challenged withHDM aerosols to induce early phase asthmatic re-sponses. At the peak of these responses (characterisedby an increase in lung resistance), the sheep were treatedfor two minutes with one of six treatment groups listedbelow. Lung resistance was monitored up to 20 minutesfollowing cessation of treatment.

1- S-1226: Nebulized perfluorooctylbromide(perflubron) generated using a Sidestream® nebulizer(Philips Respironics) and driven by a compressed gasmixture containing 12% CO2, 21% O2 and balanceN2.

2- CO2 alone: Inhalation of a gas mixture containing12% CO2, 21% O2 and balance N2.

3- Perflubron: Nebulized perflubron driven bycompressed medical air.

4- Salbutamol (1 mg/ml): Nebulized salbutamol drivenby compressed medical air.

5- Medical air: Inhalation of medical air6- No treatment: room air

Video bronchoscopyAn unsensitised sheep was used in separate experimentsto visually assess the airway response to inhaled S-1226.


Real time video recordings were taken with a broncho-scope digital camera following methacholine challenge.To insure that the same airway was recorded throughoutthe procedure, the upper region of the airway was tat-tooed by injecting tissue dye using a transbronchial nee-dle. The airway was then pre-contracted with an aerosolof methacholine (MCh) delivered via a Trudell Ltd Ptybronchoscope catheter, inserted into the biopsy port ofthe bronchoscope. Doubling doses of methacholine werenebulized directly into the lung segment (0.009, 0.018,0.037 w/v) for 30 seconds with a 2 minute interval be-tween methacholine doses until the airway was max-imally constricted. The airway was then treated withnebulized S-1226 delivered through a Trudell Pty Ltdbronchoscope catheter and the effects video recorded inreal time. If the airway re-constricted, second and thirdtreatments were administered.

Data analysisIn each experiment several intervals were analysed:Baseline, HDM-induced early phase response, and threepost-treatment periods (immediately, 1–10 minute and10 to 20 minutes after treatment cessation). Means werecalculated for each interval and treatment effect wasexpressed as percent decline of lung resistance.Analysis of variance (ANOVA) was performed over

each time interval to determine if any of the treatmentgroups performed better than any other. A two tailedDunnett’s post-hoc test was performed. This involvedseparate analyses addressing two distinct questions. Thefirst: is S-1226 superior to no treatment, medical air(negative control) or salbutamol (positive control). Sec-ondly to determine which component of S-1226, CO2 orperflubron contributed greater to its efficacy. Tests wereperformed using GraphPad Prism version 5.00 and Rversion 3.1.1 for windows. A p-value ≤ 0.05 was consid-ered significant.

Results and discussionCharacterization of nebulized perflubronResults from characterization of nebulized perflubron aresummarized in Table 1. The mean rate of perflubron deliv-ered was 598 mg/minute, of which some 203 mg/minute

Table 1 Characterization of nebulized perflubron using the S

Nebulized perflubron

Sidestreamventuri CLOSED

Nebulizer flowrate L/min

Inhaled perflubronaerosol 1 minute mg

Inhaled pVapor 1 m

Exp#1 8.5 208 392

Exp#2 8.5 198 399

Nebulized perflubron was characterized in two separate experiments (exp#1 and exnebulizer with active venturi closed. Nebulized perflubron liquid was determined topredominant physical form (means: 395 mg/min vapour vs 203 mg/min aerosol). Thand fractionated using a Marple 298X cascade impactor. The nebulized perflubrondistribution, Figure 4) having a Mass Median Aerodynamic Diameter (MMAD) of 1.1

consisted of aerosol with the remaining 395 mg/minutewas estimated to be in vapor form – thus the majority(approximately 2/3 of the nebulized perflubron) was deliv-ered in vapor form. The perflubron which was delivered inaerosol form demonstrated a relatively small particle sizedistribution, with a Mass Median Aerodynamic Diameter(MMAD) of 1.1 um, with a Geometric Standard Deviation(GSD) of 2.5 (Table 1). The perflubron aerosol size distribu-tions obtained from the Sidestream nebulizer are shown inFigure 1.The characterization of nebulized perflubron demon-

strated that the majority of the nebulized perflubronexisted in the vapor form while the remaining propor-tion of aerosol existed as a relatively fine aerosol with anMMAD of 1.1 um. We infer that perflubron as vaporand aerosol, as well as CO2 gas component of inhaled S-1226, would rapidly distribute throughout the respira-tory tract.

HDM challenge followed by treatmentSensitized and HDM challenged sheep treated with S-1226for 2 minutes during the peak of the early phase allergic re-sponse showed an immediate decline in lung resistance inall animals following HDM challenge (Figure 2).Treatment with S-1226 compared to the other arms of

the study are shown in Figure 3. Lung resistance de-clined by 50.1% ± 5.5, 47.2% ± 6.2 and 51.8% ± 8.3 (mean± SE) immediately, 1 to 10 minutes and 10 to 20 minutesafter treatment with S-1226 respectively. Treatment with12% CO2 resulted in declines in lung resistance of36.9% ± 6.8, 20.6% ± 12.0 and 24.3% ± 28.4 (mean ± SE)immediately, 1 to 10 minutes and 10 to 20 minutes aftertreatment respectively. Treatment with perflubron +medical air resulted in declines in lung resistance of24.8% ± 7.5, −1.3% ± 10.7 and −7.2% ± 5.1 (mean ± SE)immediately, 1 to 10 minutes and 10 to 20 minutes aftertreatment respectively. Treatment with salbutamol re-sulted in declines in lung resistance of 31.3% ± 6.8,15.3% ± 7.1 and 14.3% ± 9.6 (mean ± SE) immediately, 1to 10 minutes and 10 to 20 minutes after treatmentrespectively. Following inhalation of medical air, lungresistance declined by 23.1% ± 9.7, 16.0% ± 10.8 and13.1% ± 17.5 (mean ± SE) immediately, 1 to 10 minutes

idestream (venturi closed)

Aerosol size

erflubroninute mg

Inhaled Total perflubron aerosol +vapor 1 minute mg/min

MMADμm

GSD

600 1.1 2.4

597 1.1 2.6

p#2) using compressed gas flow rate of 8.5 L/min through a Sidestreampartition into aerosol and vapour states, with the vapour being thee droplet size of the aerosolized perflubron component was further examinedaerosol distribution curve showed near log normal symmetry (see normativeum, and a Geometric Standard Deviation (GSD) of 2.5.

0

5

10

15

20

25

30

35

40

0

50

100

0.1 1 10 100

No

rmat

ive

Dis

trib

uti

on

Cu

mu

lati

ve U

nd

ersi

ze %

Particle size (µm)

Figure 1 Sidestream nebulizer aerosol size distribution. Aerosol size is on the X-axis is plotted against the normative and cumulative undersideaerosol distributions. The blue boxes profile the normative size distribution of nebulized perflubron, the near symmetry suggests a log-normaldistribution of aerosol particles. The red squares plot the same data but as a cumulative size distribution of particle size against cumulative undersize%. The cumulative size distribution is used to interpolate the MMAD as the particle size at 50% of the cumulative mass – in this case the MMAD is 1.1um. The Geometric Standard Deviation (GSD) of the MMAD is calculated using the intercepts at 15.8% and 84.1% (approximated by grey lines inFigure) using the formula GSD = sq rt (size 84.1um / size 15.8 um).


and 10 to 20 minutes after treatment respectively. Lungresistance measured following HDM challenge with notreatment decreased by 1.9% ± 4.7, 24.4% ± 4.2 (mean ±SE) at 1 to 10 minutes and 10 to 20 minutes after treat-ment respectively.Inhalation of nebulized S-1226 caused a rapid and sus-

tained decrease in lung resistance during the early phaseasthmatic response which was significantly greater thanno treatment at 1–10 minutes (p = 0.002) and significantlygreater than medical air immediately, 1–10 minutes and

Figure 2 Effect of S-1226 on lung resistance in the sheep model of as9 different sheep. The treatment was delivered for 2 minutes and lung resiminutes after treatment cessation. All nine sheep showed an immediate angiven at the peak of the early phase response to inhaled allergen (HDM).

10–20 minutes after treatment (p = 0.027, 0.018 and 0.039respectively). The effect of S-1226 was significantly greaterthan salbutamol 1–10 minutes and 10–20 minutes aftertreatment (p= 0.025 and 0.046 respectively). S-1226 showeda significantly greater decline in lung resistance than perflu-bron alone immediately, 1–10 minutes and 10–20 minutesafter treatment (p= 0.019, 0.002 and 0.012 respectively). Fi-nally, the effect of S-1226 was stronger but not significantlydifferent from CO2 alone immediately, 1–10 minutes and10–20 minutes after treatment (p= 0.329, 0.124 and 0.319

thma. Changes in lung resistance following treatment with S-1226 instance was measured immediately, and at 1–10 minutes and 10–20d sustained decline in lung resistance following S-1226 treatment

Figure 3 Comparison between S-1226 and other bronchodilators in the sheep model of asthma. This figure shows the % decline in lungresistance following treatment with S-1226, 12% CO2, perflubron, salbutamol, medical air or no treatment in HDM challenged sheep. Measurementswere taken immediately, 1–10 minutes and 10–20 minutes after treatment. Treatment with S-1226 showed significant and sustained declines in lungresistance compared to medical air or no treatment. In addition S-1226 showed a significantly greater decline in lung resistance compared tosalbutamol. P-values show significant difference from S-1226; *p <0.05, # p≤ 0.01.


respectively). The CO2 decline in lung resistance appearedshorter-lasting than the decline observed with S-1226.

Video-bronchoscopy of S-1226 treatment followingMCh challengeThe rapidity of the effect was demonstrated by video-bronchoscopy. MCh challenge resulted in rapid constric-tion of the segmental and subsegmental bronchi. Withouttreatment the airway narrowing lasted for at least20 minutes before a slow natural recovery. Followingtreatment with a spray of S-1226 there was an immediatere-opening (within 5 seconds) of the MCh-constricted

Figure 4 Video bronchoscopy of the effect of S-1226. Bronchoscopic sinitiating treatment with nebulized S-1226 (arrow heads indicate tissue dye

airway (Figure 4). A video recording of this treatment ef-fect can be viewed online under Additional file 1. The ef-fect was sustained during the delivery of S-1226 and up to1 minute after treatment cessation. After that the airwaybegan to re-constrict. Second and third treatments with S-1226 had identical effects.

Potential mechanisms of actionOur study did not attempt to determine the mechanismof action of S-1226. The bronchodilatory effect of CO2

has been demonstrated in many mammalian species in-cluding cats, rats dogs [19,22] as well as sheep and

till images of sheep airways pre and post MCh and seconds after). The video presentation is available online under Additional file 1.


humans However, the literature indicates that it mightoperate through several distinct mechanisms.The first mechanism is through a direct physiologic

relaxant effect of CO2 on airway smooth muscle [20–22]. The way(s) in which CO2 regulates bronchialsmooth muscle tone is not fully understood. Recentin vitro data indicates that part of this effect is mediatedby pH dependent and part by pH independent but epi-thelium dependent mechanisms [22]. Studies in vivo in-dicate the response (in rats) to inhaled CO2 does notrequire adrenergic pathways [46]. Fisher and Hansen[27] showed in their study of exercise induced asthmathat cholinergic pathways were not involved in thebronchodilatory effects of inhaled CO2. Bronchodilatordrugs that use β- adrenergic and cholinergic pathwaysform the current basis for treating obstructive lungdisease.Inhaled carbon dioxide (5, 10 and 15%) has been shown

to reduce total lung resistance induced by intravenous his-tamine, acetylcholine and serotonin in dogs, cats and rats[19]. Inhaled CO2 has also been shown to reduce totallung resistance induced by pulmonary artery occlusion incats and dogs [19]. All species studied responded to CO2

but there were small differences in response to the differ-ent broncho-constricting agents between species.Studies by Ingram and colleagues [47] suggest that the

bronchodilatory effect of CO2 may be independent ofblood PCO2. They studied the effect of varying airwayand arterial PCO2 on airway tone in dogs. They showedthat low airway PCO2 combined with high arterial PCO2

resulted in a large increase in airway resistance [47].These findings indicate that CO2 may have unique prop-erties when delivered through the airways and supportsthe previously cited in vitro work that airway epitheliumplays a part in the relaxant response [22]. Finally therapidity of the response as seen in the sheep video indi-cates that neural mechanisms are involved. The non-adrenergic-non cholinergic (NANC) pathways are thebest candidates for this effect. The NANC system hasboth broncho-constrictive and bronchodilator compo-nents [48]. CO2 alone or S-1226 may activate thebronchodilatory pathways or inhibit the broncho-constrictive pathways. Further work is required to ad-dress these questions.The second mechanism may enhance delivery of CO2

to the airways. It could do this in several ways; first sus-tained release of CO2 dissolved in perflubron droplets asthe droplets evaporate may explain the temporal syner-gism observed with the combination treatment. Second,because much of S-1226 is in gaseous form, it will be de-livered rapidly into obstructed airways by diffusion andpossibly through collateral ventilation.The third mechanism whereby this combination treat-

ment may work is by facilitating penetration and

lubrication of mucus plugs, thus enhancing muco-ciliary clearance. We recently reported, using an in vitrosystem, that mucin plug clearance was significantly en-hanced by perflubron [49].S-1226 may also lower the surface tension in the in-

flamed airways. It has been shown that the normal air-way is lined by a thin film of endogenous surfactant witha low surface tension [50]. This film is important formaintaining small airway patency at low lung volumes[51] and is abnormal in asthma [52,53].Finally, carbon dioxide and perfluorocarbons may also

be beneficial for their anti-inflammatory properties. Per-flubron given in vitro or during partial liquid ventilationexerts an anti-inflammatory effect on alveolar cells [54],decreases white blood cell counts, [54,55] and the pro-inflammatory cytokines IL-1 and IL-6. Perflubron alsoincreases release of the anti-inflammatory chemokineIL-10 [54].

Strengths and limitations of the animal modelLike with most experimental systems, large animal modelsof allergic airway disease have both strengths, and alsolimitations, which need to be recognised when evaluatingnew therapies for asthma [56–58]. For example, clinicallyefficacious anti-asthma drugs such as leukotriene LTE4 re-ceptor antagonists, cromolyn and corticosteroids have allbeen successfully tested in the sheep model of asthma[59]. The success of these drugs is in contrast to the test-ing of platelet activated factor (PAF) antagonist therapiesin sheep. PAF antagonists were shown to alleviate allergicairway responses in sheep [60], however the positive out-come with these treatments in sheep did not translate tohumans giving negative results in clinical trials [61].Nevertheless, sheep are an appropriate species to investi-gate the efficacy of the bronchodilator reported here for anumber of important reasons. Firstly, the sheep respira-tory system is at least comparable to the human systemwith respect to lung size, lung anatomy and morphology,and the pharmacological and physiological parameters ofthe ovine lung [57–59]. Pertinent to this study, as themechanism of the bronchodilator most likely involvesboth the airway smooth muscle and the intact airway epi-thelium [22], is that the arrangement and morphology ofovine airway epithelium [62] and airway smooth muscledown the tracheo-bronchial tree [57] is functionally simi-lar to that observed in human lungs. Thus, the broncho-dilator interactions we report here for the sheep modelcould reasonably translate to the human lung. The othermajor asset of the sheep model for examining broncho-dilator effectiveness relates to the docile nature of sheep,which allows experiments to be ethically conducted inunsedated and unanaesthetised animals [59,63]. These an-aesthetic and/or sedation agents, if administered as is ne-cessary for behavioural reasons in most other animal


species modeling asthma, may otherwise dampen, andthus mask, the bronchodilator responses.In summary, we provide evidence that treatment with

inhaled S-1226 delivered as an aerosol/gas mixture mayplay an important role in the treatment of acute asthmaattacks. We show that perflubron combined with 12%CO2 is a potent airway smooth muscle relaxant in thisovine model of allergic asthma. The response to thecombination treatment was rapid and exceeded treat-ment with medical air (negative control) and salbutamol(positive control) in both amplitude and temporal com-ponents. Further work is required to fully evaluate themechanism(s) of action of this novel therapy.

Additional file

Additional file 1: Video clip description: the black dot is the stainfrom the tissue dye. The partially occluded airway that is in the centerof the screen begins to dilate immediately after the treatment starts (withthe click sound on the video). Note that the adjacent smaller, completelyoccluded, airways also re-opened with treatment.

Competing interestsSolAeroMed Inc is a small biotechnology company founded in partnershipwith the University of Calgary to develop a novel rescue drug for acuteasthma (S-1226). This paper describes pre-clinical animal studies in thedevelopment of this drug.- Dr Tamer El Mays is a co-founder, shareholder and was a temporaryemployee of SolAeroMed Inc.- Dr Richard Leigh is a shareholder in SolAeroMed Inc.- Dr Cora Pieron is a shareholder and part-time employee of SolAeroMed Inc.- Dr Dennis is a director, co-founder and shareholder in SolAeroMed Inc.- Dr Francis Green is a director, co-founder and shareholder in SolAeroMedInc.- Dr Ken Snibson is a shareholder in SolAeroMed Inc.- All remaining authors have no competing interests to declare.

Authors’ contributionsConception and Design: TE, KS, FG, RL. Sheep experiments were conductedby TE, KS, PC, EK and JV. Data acquisition and analysis by EK, JV and GS.Aerosol characterization and delivery system design by CP and JD.Manuscript drafted by TE, revisions and additions by KS, JD, CP, FG, PC, RL,EK, JV, GS. All authors read and approved the final manuscript.

Author details1Airway Inflammation Research Group, Snyder Institute for Chronic Diseases,University of Calgary, Calgary, AB, Canada. 2Centre for Animal Biotechnology,University of Melbourne, Melbourne, VIC, Australia. 3Lung Health ResearchCentre, University of Melbourne, Melbourne, VIC, Australia. 4SolAero Ltd,Calgary, AB, Canada. 5Department of Pathology & Laboratory Medicine, HRIC4AC62 3280 Hospital Drive N.W, Calgary, AB T2N 4Z6, Canada.

Received: 1 April 2014 Accepted: 13 August 2014

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doi:10.1186/s12931-014-0098-xCite this article as: El Mays et al.: Nebulized perflubron and carbondioxide rapidly dilate constricted airways in an ovine model of allergicasthma. Respiratory Research 2014 15:98.

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